Eukaryotic chromosomes are subdivided into functionally autonomous domains by special elements called chromatin boundaries or insulators. Boundaries define units of independent genetic activity by shielding genes and/or regulatory elements within a domain from adventitious interactions with regulatory elements/genes in nearby domains. The specific functions that can be ascribed to most boundary elements include an enhancer- blocking or insulator activity, a silencer-blocking or barrier activity and, when paired in appropriate combinations an ability to bring distant chromosomal DNA segments in close proximity. Genome wide experiments indicate that insulators are a ubiquitous feature of eukaryotic chromosomes from flies to man, demarcating distinct chromatin and regulatory domains and helping to mediate or restrict long-range regulatory interactions. In Drosophila insulators have been shown to play an essential role in the proper regulation of the homeotic genes in the Bithorax complex (BX-C) and we've begun a systematic analysis of several boundaries from this complex. As a starting point, we selected Fab-7. The phenotypic effects of Fab-7 mutants are well understood, and it together with its partners, the two flanking boundaries, Fab-6 and Fab-8 can be manipulated in situ. Although Fab-7 is seemingly active throughout development, this constitutive activity is generated by cis-acting elements/trans-acting factors whose activity is developmentally restricted. Our approach is to analyze the proteins that interact with and are critical for the functioning of the Fab-7 boundary, its two neighbors and a special element, Abd-BPR associated with the Abd-B promoter using a combination of biochemistry, cell biology and genetics. We've identified three new stage specific Fab-7 boundary factors, Elba, Insv, and LBC, which are shared with Fab-8 and likely also with Fab-6 and Abd-BPR. These factors are large multi-protein complexes ranging in size from ~240-650 kD and they have unusual DNA binding properties. For example, the minimal LBC recognition sequence is ~65 bp; however, the actual in vivo substrates for this complex may be several hundred bp in length. The Elba and Insv insulator complex proteins both have a conserved BEN DNA binding domain. Mammals have a large family of genes encoding BEN domain proteins and they have been implicated in embryonic stem cell pluripotency, neurogenesis, cocaine addiction and cancer. Based on what we now know about the fly BEN domain proteins, it is likely that most all of these mammalian proteins will have insulator functions that are stage, tissue or cell type specific. This proposal describes studies focused on characterizing the fly complexes in detail, determining how they function to confer insulator activity, understanding how they contribute to the blocking and bypass activities of the insulators in the Abd-B TARD that are critical for the proper parasegment specific regulation of the Abd-B gene, and finally exploring their roles in the development and differentiation of the fly gonad.